NADPH and NADH are important redox mediators in organisms. Although the difference of their chemical structure is only one phosphate group, their biological function is significantly different. NADH participates in decomposition reactions and plays an important role in cellular respiration and nutrient metabolism, whereas, NADPH participates in synthetic reactions and is consumed to provide energy for biosynthesis of large molecules. NADPH also plays an important role in cellular defensive oxygen (ROS) injury as a vital component of the intracellular antioxidant defense system. Therefore, accurate quantification of the concentration of NADPH and NADH is important for the detection of biological actions and the diagnosis of diseases.
NADPH and NADH can be detected by the color generated in their redox reactions with tetrazoliun salts. The TTC (2, 3, 5-triphenyltetrazolium salt) was first synthesized in 1894 and is a fat-soluble light-sensitive compound for the detection of seed viability. The MTT assay is another method for detecting cell survival and growth. MTT is reduced to—water-insoluble blue-violet crystalline—formazan by succinate dehydrogenase from living cellular mitochondria, then absorbance can be detected under 540 nm or 720 nm after the formazan is dissolved by DMSO. The assay has advantages of high sensitivity, favorable economic benefit and so forth, but the disadvantage is the need for multiple steps in the detection process. Water-soluble WST (bisulfonic phenyltetrazolium salt) was synthesized in 1997 and used in commercial assays for cell viability. However, water-soluble WST-8 would not be easily synthesized and purified and its purity and quality are difficult to control.